CN116751773A - D-psicose 3-epimerase mutant, gene and application - Google Patents
D-psicose 3-epimerase mutant, gene and application Download PDFInfo
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- 108030002106 D-psicose 3-epimerases Proteins 0.000 title claims abstract description 65
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 20
- 150000001413 amino acids Chemical class 0.000 claims abstract description 26
- 235000001014 amino acid Nutrition 0.000 claims abstract description 21
- 229940024606 amino acid Drugs 0.000 claims abstract description 21
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 10
- 229960000310 isoleucine Drugs 0.000 claims abstract description 10
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims abstract description 10
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims abstract description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000004474 valine Substances 0.000 claims abstract description 8
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 6
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 6
- 239000004220 glutamic acid Substances 0.000 claims abstract description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 4
- 235000003704 aspartic acid Nutrition 0.000 claims abstract description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 102000004190 Enzymes Human genes 0.000 claims description 26
- 108090000790 Enzymes Proteins 0.000 claims description 26
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 claims description 20
- 230000035772 mutation Effects 0.000 claims description 18
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- 238000012408 PCR amplification Methods 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 229910001429 cobalt ion Inorganic materials 0.000 claims description 4
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
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- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 6
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 6
- 238000006555 catalytic reaction Methods 0.000 description 5
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
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- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- 239000004475 Arginine Substances 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 150000000837 D-psicose derivatives Chemical class 0.000 description 1
- 108030002100 D-tagatose 3-epimerases Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101710094530 L-ribulose 3-epimerase Proteins 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000961103 [Clostridium] bolteae ATCC BAA-613 Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
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- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
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- 239000002894 chemical waste Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
- 238000012900 molecular simulation Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
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- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
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Abstract
The application belongs to the technical field of bioengineering, and particularly relates to a D-psicose 3-epimerase mutant, a gene and application thereof, wherein the mutant is obtained by mutating 265 th amino acid or 265 th and 268 th amino acid on the basis of a D-psicose 3-epimerase sequence shown in SEQ ID NO. 1; the 265 th amino acid is changed from isoleucine to valine; alternatively, the amino acid at position 265 is changed from isoleucine to valine and the amino acid at position 268 is changed from glutamic acid to aspartic acid; the application can improve the catalytic activity of the D-psicose 3-epimerase.
Description
Technical Field
The application belongs to the technical field of bioengineering, and particularly relates to a D-psicose 3-epimerase mutant, a gene and application.
Background
D-psicose is epimer of D-fructose at C-3 position, and is rare in natural content, sweetness is 70% of that of sucrose, and energy is only 0.3%. Meanwhile, D-psicose is rated as "GRAS" safety class by the united states food and drug administration, and is proposed as an ideal substitute for sucrose in foods. In recent years, studies have shown that D-psicose has the ability to lower blood glucose levels, prevent atherosclerosis, and scavenge Reactive Oxygen Species (ROS), and thus has received increasing attention. In addition, some derivatives of D-psicose have anticancer and antiviral activities. Therefore, the efficient production of D-psicose shows a broad market prospect.
The D-psicose 3-epimerase can carry out isomerization reaction through a C3-O3 proton exchange mechanism to biosynthesize D-psicose from D-fructose. Currently, studies on biosynthesis of D-psicose from D-fructose using D-psicose 3-epimerase have been greatly progressed based on the Izumorin isomerization strategy. D-psicose 3-epimerase belongs to the DTEases family. The enzymes of the DTEases family are classified into three types of D-tagatose 3-epimerase, D-psicose 3-epimerase and L-ribulose 3-epimerase, respectively, according to their optimal substrates. Since the most suitable substrate for D-psicose 3-epimerase is D-psicose, it is the isomerase most commonly used at present for the production of D-psicose.
The production method of D-psicose mainly comprises natural extraction, chemical synthesis and biosynthesis, and biosynthesis is the main production mode at present. D-psicose is slightly contained in nature, and is difficult to synthesize in large quantities by natural extraction. The production of D-psicose by chemical synthesis requires a complex and expensive separation process, generates a large amount of chemical waste and byproducts, and has a negative effect on the environment. In contrast, enzyme catalysis has the characteristics of high catalytic efficiency and specificity, low energy consumption and environmental protection, and development of the D-psicose 3-epimerase which is efficient, high in stability and easy to prepare is necessary. At present, D-psicose 3-epimerase resources from various sources are collected, but the environment hypochondriac force existing in industrial production makes the production process for producing D-psicose by natural enzyme catalysis not efficient and applicable. The research shows that the genetic engineering can effectively improve the stability, the catalytic activity and various enzymatic properties of the protein, including the replacement of promoters, signal peptides, protein engineering and the like. In industrial production, the catalytic activity of the existing D-psicose 3-epimerase needs to be further improved so as to realize the efficient production of the D-psicose by single-enzyme catalytic synthesis.
Disclosure of Invention
The application aims to provide a D-psicose 3-epimerase mutant, a gene and application thereof, and the D-psicose 3-epimerase mutant is used for improving the catalytic activity of D-psicose.
The application provides a D-psicose 3-epimerase mutant, which is obtained by mutating 265 th amino acid or 265 th and 268 th amino acid on the basis of a D-psicose 3-epimerase sequence shown in SEQ ID NO.1 (NCBI accession number A8RG82.1);
the 265 th amino acid is changed from isoleucine to valine (I265V), and the amino acid sequence of the 265 th amino acid is SEQ ID NO.2; or alternatively, the process may be performed,
the 265 th amino acid is changed from isoleucine to valine, the 268 th amino acid is changed from glutamic acid to aspartic acid (I265V/E268D), and the amino acid sequence is SEQ ID NO.3.
The nucleotide sequence of the D-psicose 3-epimerase shown in SEQ ID NO.1 (NCBI accession number A8RG82.1) is SEQ ID NO.4.
The application also provides a preparation method of the D-psicose 3-epimerase mutant, which comprises the following steps:
a. determining 265 th amino acid as mutation site based on amino acid sequence of wild D-psicose 3-epimerase in sequence table SEQ ID NO.1, and designing mutation primer of site-directed mutation:
the site-directed mutagenesis primer for introducing the I265V mutation is as follows:
forward primer I265V-F:5'-GGTAGTGAGATTAAAGTATGGCGTG-3' (SEQ ID NO. 7),
reverse primer I265V-R:5' -TACTTTAATCTCACTACCAACTGTACCACCTTGCATTACGAA-3' (underlined as mutant base) (SEQ ID NO. 8).
The site-directed mutagenesis primer for introducing the I265V/E268D mutation is:
forward primer I265V/E268D-F:5'-ATTAAAGTATGGCGTGATATGGTTCC-3' (SEQ ID NO. 9),
reverse primer I265V/E268D-R:
5’-ATCACGCCATACTTTAATATCACTACCAACTGTACCACCTTGCATTACGAA-3' (underlined as mutant base) (SEQ ID NO. 10).
b. C, carrying out PCR amplification by using a vector (preferably pET32a (+)) as a template and using the primer in the step a, and recovering a PCR product rubber cutting;
c. and c, transforming the vector obtained in the step b into a host cell to obtain D-psicose 3-epimerase mutants, namely mutant I265V and mutant I265V/E268D.
Preferably, the carrier pET32a (+) in the step b is pET32a (+) plasmid containing D-psicose 3-epimerase gene is subjected to BamH I and HindIII double digestion, and meanwhile, the obtained D-psicose 3-epimerase gene fragment is subjected to rubber cutting recovery and then is connected to the cut pET32a (+) plasmid through seamless cloning.
Preferably, the vector in the step b is any one of a PUC series, a PET series or a PGEX series, and more preferably, the vector in the step b is a PET series plasmid vector.
The application provides an enzyme preparation, which comprises the D-psicose 3-epimerase mutant. The enzyme preparation of the present application may further contain cobalt ions (e.g., cobalt chloride) and buffers such as sodium dihydrogen phosphate and disodium hydrogen phosphate buffers.
The application provides a gene for encoding the D-psicose 3-epimerase mutant.
Preferably, the nucleotide sequence of the gene is SEQ ID NO.5 or SEQ ID NO.6.
The present application provides an expression vector comprising the gene.
The application provides a recombinant bacterium containing the expression vector.
Preferably, the host cell of the recombinant bacterium may be a fungal cell or a bacterial cell, preferably a gram positive or gram negative bacterial cell, more preferably E.coli, preferably BL21 (DE 3).
The application provides an application of the D-psicose 3-epimerase mutant or the recombinant bacterium for converting a substrate into D-psicose.
Preferably, the substrate is fructose; during the conversion process, metallic cobalt ions are added.
Preferably, the fructose is D-fructose, the concentration of the metal cobalt ion is 1mM, and the pH value of the conversion is neutral.
The sequences of SEQ ID NOS.1-6 according to the present application are shown in tables 1 and 2.
TABLE 1 amino acid sequence listing
TABLE 2 nucleotide sequence listing
The application has the beneficial effects that the amino acid in the active center of the D-psicose 3-epimerase is determined through molecular docking simulation, and the amino acid residue near the active site of the protein molecule is changed through rational design mutation, so that the catalysis efficiency of the D-psicose 3-epimerase is improved, and the yield of the D-psicose 3-epimerase is further improved.
The D-psicose 3-epimerase secreted by the recombinant escherichia coli constructed by the application can improve the enzyme activity of the D-psicose 3-epimerase by 1.51 and 1.00 times compared with that of a starting strain. The mutant enzyme activity of the transformed genetically engineered bacteria is obviously improved, the genetically engineered bacteria are more suitable for industrial application, the production cost can be reduced, and the production efficiency is improved.
Compared with wild type or other mutated isomerase, the D-psicose 3-epimerase constructed by the application has higher enzyme activity and obviously improves economic benefit.
Drawings
FIG. 1 is a diagram showing the electrophoresis of a nucleic acid gel of D-psicose 3-epimerase after mutation;
m is marker;1 is the PCR amplification product of recombinant plasmid I265V, 2 is the amplification product of recombinant plasmid I265V/E268D.
FIG. 2 is a gel electrophoresis of D-psicose 3-epimerase protein in a mutant strain;
m is marker,1 is wild type protein gel electrophoresis lane, 2 is protein gel electrophoresis lane of mutated isomerase I265V, 3 is protein gel electrophoresis lane of mutated isomerase I265V/E268D.
FIG. 3 shows the relative enzyme activities of D-psicose 3-epimerase having the 265 locus mutation, 265 locus mutation and 268 locus mutation;
Cb-WT is wild-type D-psicose 3-epimerase, cb-I265V is 265 site mutated D-psicose 3-epimerase, cb-I265V/E268D is 265 site and 268 site mutated D-psicose 3-epimerase.
Detailed Description
Further description will be provided below in connection with specific examples. The technical means used in the examples are conventional means well known to those skilled in the art, if specifically indicated.
The raw materials and reagents used in the examples of the application are all conventional chemical reagents and can be purchased commercially. LB medium was prepared as follows: 5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride.
Determination of the enzyme Activity of D-psicose 3-epimerase:
d-fructose with the concentration of 50g/L is taken as a catalytic substrate, a proper amount of purified enzyme solution is added, and the reaction is carried out at 1mM Co 2 + Immediately after 15min of catalysis at 55℃and pH 7.0, the reaction was stopped by a boiling water bath for 10min. Subsequently, the whole system was centrifuged at 12000r/min for 10min and then treated with 0.22. Mu.mThe filtrate can be detected by High Performance Liquid Chromatography (HPLC).
The HPLC detection conditions were as follows: chromatographic column Carbomix-Pb-NP10:8% (7.8X100 mm), mobile phase of ultrapure water, flow rate of 0.5mL/min, column temperature of 78 ℃, differential (RI) detector, D-fructose standard retention time of 20.12min, D-psicose standard retention time of 34.13min.
Definition of enzyme activity unit (U): the amount of enzyme required to produce 1. Mu. Mol of D-psicose per unit time under standard conditions.
Example 1: d-psicose 3-epimerase crystal structure simulation
The crystal structure of the Clostridium bolteae ATCC BAA-613-derived D-psicose 3-epimerase was simulated using molecular simulation docking software using the reported Clostridium cellulolyticum H D-psicose 3-epimerase (PDB code:3 VNI) as a template (crystal structure of D-psicose 3-epimerase from Clostridium cellulolyticum H10and its complex with ketohexose sugars, published 2012) (amino acid similarity of 53.1%). After the 3D structure of the protein in example 1 was simulated for docking, glutamic acid 265 and arginine 268 near the catalytic domain of D-psicose 3-epimerase were selected as the active related sites of the D-psicose 3-epimerase of the present study.
Example 2
Influence of the mutation of the site related to the activity of the D-psicose 3-epimerase on the expression of the D-psicose 3-epimerase
The 265 th, 265 th and 268 th amino acids are mutated by utilizing a fixed point, primers I265V-F, I265V-R and I265V/E268D-F, I V/E268D-R are designed, PCR amplification is carried out by taking the constructed pET32a (+) Cb-DAE as a template, and the 265 th isoleucine is mutated and replaced, or the 265 th isoleucine and 268 th glutamic acid are mutated, and the PCR reaction conditions are as follows: 98℃for 5min,28 cycles (98℃for 10s, 56℃for 30s, 72℃for 1 min) and 72℃for 10min. PCR amplification system: 2. Mu.L of template, 2. Mu.L of upstream and downstream primers, 25. Mu.L of Prime Star (Premix) DNA, ddH 2 O19. Mu.L. And purifying and recovering the PCR product by using a gel recovery kit, and detecting the concentration of the recovered product by electrophoresis. Carrier inThe enzyme digestion is carried out under the action of BamHI and HindIII fast cutting enzymes, the PCR recovery product is connected by a seamless cloning method, the PCR recovery product is transformed into competent E.coil BL21 (DE 3), ampicillin LB plates are coated, and positive colonies are picked. Plasmid was extracted after shaking overnight culture at 37℃to obtain site-directed mutant strains.
In example 2, when the 265 th isoleucine point of the genetically engineered D-psicose 3-epimerase is mutated into valine, the enzyme activity of the D-psicose 3-epimerase is obviously improved (as shown in figure 3) and is 2.51 times that of the original strain, named pET32a (+) Cb-I265V, and then the E.coli BL21 (DE 3) is transferred to obtain a mutant strain E.coli BL21 (DE 3) pET32a (+) Cb-I265V with site-directed mutagenesis. It was demonstrated that the enzyme activity of the D-psicose 3-epimerase was significantly altered by point mutation at position 265.
In example 2, when the 265 th isoleucine point of the genetically engineered D-psicose 3-epimerase is mutated into valine and the 268 th glutamic acid is mutated into aspartic acid, the enzyme activity of the D-psicose 3-epimerase is obviously improved (as shown in figure 3), which is 2.00 times that of the original strain, named pET32a (+) Cb-I265V/E268D, and then the E.coli BL21 (DE 3) is transferred to obtain a mutant strain E.coli BL21 (DE 3) pET32a (+) Cb-I265V/E268D with site-directed mutagenesis. It was demonstrated that the enzyme activity of the D-psicose 3-epimerase was significantly altered by point mutation at positions 265 and 268.
Example 3: expression purification of D-psicose 3-epimerase mutants
Inoculating the mutants I265V, I V/E268D into LB liquid culture medium (containing 100mg/L ampicillin) respectively for 8h, and inoculating the mutants into LB liquid fermentation medium (containing 100mg/L ampicillin) respectively according to 1% of inoculum size for culture; culturing at 37deg.C for 2.5 hr, respectively adding 0.25mmol/L final concentration of IPTG (isopropyl thiobeta D-galactoside) for induction, culturing at 20deg.C for 24 hr, centrifuging 50mL of fermentation broth at 4deg.C and 12000rpm for 10min, discarding supernatant, collecting thallus, precipitating with 50mmol/L, pH 7.0, na 2 HPO 4 NaH 2 PO 4 The buffer solution is resuspended and mixed well. Then fine by ultrasonic waveThe cell wall of the thallus is crushed by the cyto crusher (working condition of the ultrasonic cyto crusher: working time is 15min, working power is 40% after 2s is stopped for 3 s), and crude enzyme liquid is obtained after crushing. Subsequently, ni is utilized 2+ And purifying by an affinity column to obtain the high-purity protein.
Example 4: ability of conversion to D-psicose
The wild type D-psicose 3-epimerase enzyme solution and the mutant I265V, I V/E268D enzyme solution obtained in example 3 were added to a concentration of 50g/L of D-fructose as a catalytic substrate at 1mM Co 2+ The catalytic reaction was carried out at pH 7.0, and immediately after 15min, the reaction was stopped by boiling water bath for 10min. Subsequently, the whole system was centrifuged at 12000r/min for 10min, and then filtered with a 0.22 μm filter membrane, and the filtrate was detected by High Performance Liquid Chromatography (HPLC), and the detection results are shown in Table 1.
TABLE 1 relative enzyme Activity of different enzyme solutions at different temperatures
| Reaction temperature | Wild type | I265V | I265V/E268D |
| 45℃ | 100％ | 249.73％ | 197.42％ |
| 50℃ | 100％ | 246.85％ | 199.39％ |
| 55℃ | 100％ | 251.46％ | 200.22％ |
| 60℃ | 100％ | 247.63％ | 190.57％ |
| 65℃ | 100％ | 239.20％ | 195.72％ |
The results are shown in Table 1, and the mutant showed significantly improved enzyme activity at 55℃as compared with the wild-type enzyme. Therefore, the mutant can improve the D-psicose production under the same conditions, and the D-psicose 3-epimerase mutant can obviously improve the enzyme activity and the catalytic activity, and has good industrial application prospect.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of protection of the application is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the application, the steps may be implemented in any order and there are many other variations of the different aspects of one or more embodiments of the application as described above, which are not provided in detail for the sake of brevity.
One or more embodiments of the present application are intended to embrace all such alternatives, modifications and variations as fall within the broad scope of the present application. Accordingly, any omissions, modifications, equivalents, improvements and others which are within the spirit and principles of the one or more embodiments of the application are intended to be included within the scope of the application.
Claims (10)
1. A D-psicose 3-epimerase mutant is characterized in that the mutant is obtained by mutating 265 th amino acid or 265 th and 268 th amino acid on the basis of a D-psicose 3-epimerase sequence shown in SEQ ID NO. 1;
the 265 th amino acid is changed from isoleucine to valine; or alternatively, the process may be performed,
the 265 th amino acid is changed from isoleucine to valine, and the 268 th amino acid is changed from glutamic acid to aspartic acid.
2. A method for preparing the D-psicose 3-epimerase mutant according to claim 1, comprising the steps of:
a. determining 265 th amino acid as mutation site based on the amino acid sequence of D-psicose 3-epimerase shown in SEQ ID NO.1, and designing mutation primer of site-directed mutation:
the site-directed mutagenesis primer for introducing the I265V mutation is as follows:
forward primer I265V-F:5'-GGTAGTGAGATTAAAGTATGGCGTG-3' the number of the individual pieces of the plastic,
reverse primer I265V-R:5' -TACTTTAATCTCACTACCAACTGTACCACCTTGCATTACGAA-3’；
The site-directed mutagenesis primer for introducing the I265V/E268D mutation is:
forward primer I265V/E268D-F:5'-ATTAAAGTATGGCGTGATATGGTTCC-3' the number of the individual pieces of the plastic,
reverse primer I265V/E268D-R:
5’-ATCACGCCATACTTTAATATCACTACCAACTGTACCACCTTGCATTACGAA-3’；
b. c, carrying out PCR amplification by taking the carrier as a template and carrying out site-directed mutagenesis in the step a;
c. and c, transforming the vector obtained in the step b into a host cell to obtain the D-psicose 3-epimerase mutant.
3. An enzyme preparation comprising the D-psicose 3-epimerase mutant according to claim 1.
4. A gene encoding the D-psicose 3-epimerase mutant according to claim 1.
5. The gene according to claim 4, wherein the nucleotide sequence of the gene is SEQ ID NO.5 or SEQ ID NO.6.
6. An expression vector comprising the gene of claim 4 or 5.
7. A recombinant bacterium comprising the expression vector according to claim 6.
8. The recombinant bacterium according to claim 7, wherein the host cell of the recombinant bacterium is E.coli.
9. Use of a mutant D-psicose 3-epimerase according to claim 1 or a recombinant bacterium according to claim 7 for converting a substrate into D-psicose.
10. The use according to claim 9, wherein the substrate is fructose; during the conversion process, metallic cobalt ions are added.
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